This paper describes variation at 6 microsatellite loci in 107 specimens of Ctenomys minutus from a chromosomal hybrid zone on the coastal plain of southern Brazil. Of the 56 alleles uncovered in this study, 39.2% are exclusive to alternative cytotypes or the contact populations. Clinal variation is not obvious because there are no microsatellite fixed differences among chromosomally divergent populations, but variation at Hai 2 locus is gradual across the zone. The local populations are highly differentiated and structured. The moderate estimated values of gene flow follow an isolation-by-distance model, which predicts concentration of an allele or a homozygous genotype in patches with generations of individuals with limited dispersal and mating by proximity. Analysis of populations distantly localized from the contact zone probably will provide further insights in the genetic relationship among cytotypes.